In cytodiagnostics and in pathology, stained samples are analyzed by means of a microscope, generally in transmitted light bright field illumination. The color of the microscopically analyzed sample is an important criterion for diagnosis. In other microscopic analyses, for example by contrast methods such as phase contrast or differential interference contrast (DIC), the color of the sample is of less significance. In contrast methods of this type, unstained samples are analyzed, and appear predominantly transparent in transmitted light bright field microscopy. The contrast methods are subsequently used to make phase properties of the sample visible.
Fluorescence microscopy is a further known analysis method. In this context, the sample which is to be analyzed is illuminated by means of an incident light illumination beam path, which passes through what is known as an excitation filter. The excitation light leads to fluorescences in the object which is stained with fluorochromes, the radiated fluorescence light determining the resulting microscope image of the sample. These microscopy methods have been known per se for a relatively long time. For further details, reference is made to the available prior art.
In the last few decades, halogen lamps have been used as the illumination means in the microscope, for example for transmitted light bright field illumination. The light which is emitted by the halogen lamp predominantly corresponds to the continuous spectrum of a black body. Generally, a thermal protection filter is also built into a lighting module comprising a halogen lamp, and greatly attenuates the infra-red range of the emitted radiation. An absorption glass (KG1 having 2 mm thickness) is often used as a thermal protection filter. The continuous spectrum of the resulting illumination makes a reliable color assessment by the user possible.
In the case of illumination with a particular light source, what is known as the color rendering index (CRI) is of importance for assessing colors. This is understood to be a photometric value which can be used to describe the quality of the color rendering of light sources of equal correlated color temperature. Up to a color temperature of 5000 K, the light emitted from a black body of the corresponding color temperature is used as a reference for assessing the rendering quality. Above a color temperature of 5000 K, a daylight-like spectral distribution is used as a reference. For example, for calculating the color rendering of a household filament bulb, which is itself a good approximation to a black body, the spectrum of a black body having a temperature of 2700 K is used. Any light source which perfectly imitates the spectrum of a black body of equal (correlated) color temperature in the range of the visible wavelengths achieves a color rendering index of 100. Halogen lamps, similarly to filament bulbs, can achieve color rendering indices of up to 100.
In microscopy, the halogen lamp is increasingly being replaced with light-emitting diodes (LEDs in the following), which have known advantages. These advantages include greater light radiation at a lower consumption of electrical power and a longer service life. For transmitted light illumination, white LEDs are predominantly used. In a white standard LED, a blue, violet or UV LED is combined with photoluminescent material. Use is generally made of a blue LED, which is combined with a yellow luminescent material. UV LEDs comprising a plurality of different luminescent materials (generally red, green and blue) may also be used. In accordance with the principles of additive color mixing, white light is produced by LEDs of this type. The components manufactured in this manner have good color rendering properties, the color rendering indices being between 70 and 90. However, the white LEDs do not emit a continuous spectrum. White light LEDs which are based on blue LEDs have a strong emission in the blue spectral range (at approximately 450 nm), a minimum in the blue-green (at approximately 500 nm) and a wider emission range up to higher wavelengths, with a maximum at approximately 550 nm, which decreases considerably at approximately 650 nm.
Depending on the type of LED, the ratio of the intensity minimum at 500 nm to the intensity maximum at approximately 450 nm is typically approximately 10-20%. With a discontinuous spectrum of this type as the sample illumination, the color assessment is more difficult, and differs from the empirical values obtained in the case of microscope illumination by means of a halogen lamp.
A specific problem occurs in fluorescence microscopy, which was addressed in the introduction above. If, in the case of the transmitted light bright field microscopy which was discussed above, there is also the possibility of fluorescence microscopy, the inventors found the following effect. A large proportion of the excitation light for the fluorescence excitation passes through the sample and reaches the transmitted light illumination source along the transmitted light illumination axis. If a white light LED is used in this context, blue excitation light leads to excitation of the yellow-green conversion dye in the white LED, in such a way that, in turn, yellow-green light reaches the sample along the transmitted light illumination axis. This is perceived as a disruptive background in the fluorescence image, and can overlap considerably with the actual fluorescence from the sample. This effect occurs even when the white light LED is switched off.
The object of the present invention is therefore to improve the analysis of a sample by means of a microscope in transmitted light bright field illumination or in incident light fluorescence illumination, and to reduce the disruptive radiation in the fluorescence image.